Dihydroxy-acid dehydratase (DHAD), also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate and of 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. The DHAD enzyme, classified as E.C. 4.2.1.9, is part of naturally occurring biosynthetic pathways producing valine, isoleucine, leucine and pantothenic acid (vitamin B5). Increased expression of DHAD activity is desired for enhanced microbial production of branched chain amino acids or of pantothenic acid.
DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is also a common step in the multiple isobutanol biosynthetic pathways that are disclosed in commonly owned and co-pending US Patent Application Publication US 20070092957 A1. Disclosed therein is engineering of recombinant microorganisms for production of isobutanol. Isobutanol is useful as a fuel additive, whose availability may reduce the demand for petrochemical fuels.
For improved production of compounds synthesized in pathways including dihydroxy-acid dehydratase, it is desirable to express a heterologous enzyme that provides this enzymatic activity in the production host of interest. Obtaining high functional expression of dihydroxy-acid dehydratases in a heterologous host is complicated by the enzyme's requirement for an Fe—S cluster, which involves availability and proper loading of the cluster into the DHAD apo-protein.
Fe—S cluster requiring DHAD enzymes are known in the art and are found either in the [4Fe-4S] or [2Fe-2S] form. Some bacterial enzymes are known, the best characterized of which is from E. coli (Flint, D H, et al. (1993) J. Biol. Chem. 268:14732-14742). However these bacterial enzymes are all in the [4Fe-4S] form. The only [2Fe-2S] form reported to date is a spinach enzyme (Flint and Emptage (1988) J. Biol. Chem. 263:3558-3564).
It is desirable to use the [2Fe-2S] form of the enzyme in host cells to enhance the production of introduced biosynthetic pathways as the [2Fe-2S] form creates a lesser burden on Fe—S cluster synthesis and/or assembly. Unfortunately, only one [2Fe-2S] form of this enzyme is known.
There exists a need therefore to identify new [2Fe-2S] forms of DHAD for use in recombinant host cells where the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is a metabolic pathway step in a desired biosynthetic pathway.